Experimental Cell Research 349 (2016) 282–290 Contents lists available at ScienceDirect Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr PDGF-A and PDGF-B induces cardiac fibrosis in transgenic mice crossmark⁎ Radiosa Gallinia,b, Per Lindblomc,1, Cecilia Bondjersc,2, Christer Betsholtza,b, Johanna Andraeb, a Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden b Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden c Department of Medical Biochemistry, Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden ARTICLE INFO ABSTRACT Keywords: Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) contribute to normal heart development. PDGF Deficient or abnormal expression of Pdgf and Pdgfr genes have a negative impact on cardiac development and Transgene function. The cellular effects of PDGFs in the hearts of Pdgf/Pdgfr mutants and the pathogenesis of the Myosin heavy chain resulting abnormalities are poorly understood, but different PDGF isoforms induce varying effects. Here, we Fibrosis generated three new transgenic mouse types which complete a set of studies, where all different PDGF ligands Heart have been expressed under the same heart specific alpha-myosin heavy chain promoter. Transgenic expression of the natural isoforms of Pdgfa and Pdgfb resulted in isoform specific fibrotic reactions and cardiac hypertrophy. Pdgfa overexpression resulted in a severe fibrotic reaction with up to 8-fold increase in cardiac size, leading to lethal cardiac failure within a few weeks after birth. In contrast, Pdgfb overexpression led to focal fibrosis and moderate cardiac hypertrophy. As PDGF-A and PDGF-B have different affinity for the two PDGF receptors, we analyzed the expression of the receptors and the histology of the fibrotic hearts. Our data suggest that the stronger fibrotic effect generated by Pdgfa overexpression was mediated by Pdgfrα in cardiac interstitial mesenchymal cells, i.e. the likely source of extracellular matrix depostion and fibrotic reaction. The apparent sensitivity of the heart to ectopic PDGFRα agonists supports a role for endogenous PDGFRα agonists in the pathogenesis of cardiac fibrosis. 1. Introduction multiple cellular functions, such as cell proliferation, differentiation, cytoskeletal rearrangements and cell migration including chemotaxis. Cardiac fibrosis is characterized by excessive production of extra- In normal vertebrates, members of the PDGF family are widely cellular matrix proteins such as collagens and fibronectin deposited by expressed throughout the body and play roles both during organogen- activated fibroblasts (a.k.a. myofibroblasts). These cells accumulate at esis and during disease processes. To-date, four PDGF ligands have sites of injury or inflammation in response to locally released fibrogenic been identified (PDGF-A, -B, -C and -D), which form four homodimers mediators. The origin of cardiac myofibroblasts is unclear but may (AA, BB, CC and DD) and one heterodimer (AB) that bind to and potentially involve multiple sources, such as cardiac fibroblasts, activate two different tyrosine kinase receptors (PDGFRα and -β) with fibroblast progenitors, vascular mural cells, epicardial epithelium and different affinity. A wide variety of potential ligand-receptor interac- endothelial cells (reviewed by [1,2]). Accumulation of extracellular tions have been demonstrated in vitro, but not all have been confirmed matrix proteins in the cardiac interstitium causes myocardial stiffness in developmental in vivo studies of knockout mice (reviewed by [5]). In and ventricular dysfunction. Organ failure due to fibrosis is indeed the general, PDGF-A and -C bind to PDGFRα, and PDGF-B and -D bind to major cause of death from inflammatory diseases. Unfortunately, PDGFRβ in vivo. therapies directly targeting fibrosis or its pathogenesis are still limited All PDGFs have been reported to influence heart development. (reviewed by [3,4]). Endothelial cells express PDGF-B and -D, whereas vascular mural cells Several molecular mediators are active during cardiac fibrosis, one (smooth muscle cells and pericytes) express PDGFRβ. Genetic loss-of- of them being the platelet-derived growth factors (PDGFs). PDGF function of PDGF-B or PDGFRβ in mice lead to a hypoplastic signalling has been implicated in fibrosis of different organs, such as myocardium that lack vascular smooth muscle cells [6,7], whereas lung, liver, skin, kidney and heart (reviewed by [5]). PDGFs affect deletion of PDGF-D causes only a mild vascular phenotype in the heart ⁎ Corresponding author. E-mail address: [email protected] (J. Andrae). 1 Current address: AstraZeneca R & D, Mölndal, Sweden. 2 Current address: Sahlgrenska University Hospital, Gothenburg, Sweden. http://dx.doi.org/10.1016/j.yexcr.2016.10.022 Received 12 October 2016; Received in revised form 26 October 2016; Accepted 27 October 2016 Available online 02 November 2016 0014-4827/ © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by/4.0/). R. Gallini et al. Experimental Cell Research 349 (2016) 282–290 [8]. PDGF-A and -C are both expressed by myocardial cells, whereas discomfort in experimental animals and an hypothesis for assessment. PDGFRα-positive interstitial cells have been identified in the epicar- [22]; Recognizing and assessing pain, suffering and distress in labor- dium, myocardium and endocardium. PDGFRα signalling is needed artory animals: a survey of current practice in the UK with recommen- during the establishment of the second heart field-derived structures, dations, P. Hawkins, Laboratory Animals (2002); 36. such as ventricular septum, epicardial cells, epicardial-derived fibro- blasts, second heart field-derived myocardium, epicardial-mesenchy- 3. Generation of transgenic mice mal derivates, cardiac neural crest cells, sinus venosus and outflow tract [6,9–13]. Transgenic mice were produced by pronuclear injection of the DNA Several reports point to a surprising complexity in the cardiac constructs schematically outlined in Fig. 2. The α-MHC promoter [23] responses to PDGF/PDGFR signalling, suggesting that fibrogenic was cloned together with the full cDNA clones for PDGF-Ashort (clone responses to PDGFs may be both model- and context-dependent. A 13.1, [24]), PDGF-Along (clone D1, [25]) and PDGF-B [26]. DNA primary general increase in PDGFRα activation in mice leads to multi- constructs were excised from the vector backbone, purified using the organ fibrosis, including in the heart [14], whereas increased PDGFRβ Qiaex II gel extraction kit (Qiagen) and injected into fertilized C57BL6/ activation does not [15]. In a heterotypic heart transplantation model CBA oocytes, which were subsequently cultured until two-cell stage, in rats, administration of adenoviruses expressing PDGF-A, -C and -D and transplanted into pseudo-pregnant B6 females. For screening/ led to accelerated cardiac fibrosis and chronic rejection [16]. Likely, genotyping by Southern Blot, tail biopsies were lyzed in 500 μl lysis myocardial injection of adenoviruses expressing different PDGFs buffer (50 mM Tris, pH 8; 100 mM EDTA; 100 mM NaCl; 25 μl 20% results in an increased inflammatory reaction through PDGFRα SDS and 25 μl 10 mg/ml proteinase K) and DNA was purified by activation. Moreover, PDGFRα and -β neutralizing antibodies were phenol/chloroform extraction and ethanol precipitation. Southern blot shown to attenuate the response to myocardial infarction, including was performed with standard techniques using PDGF-A and PDGF-B decreased collagen deposition and impaired neovessel maturation [17]. human cDNA as probes [25,27,28]. By administrating adenoviruses expressing different PDGF isoforms to the heart of adult mice, we recently described that whereas PDGF-B 4. Genotyping of mice aggravated the adenovirus-induced inflammation PDGF-D attenuated it, suggesting that different modes of activation of the same receptor For PCR genotyping of transgenic founders, tail biopsies were lysed may result in seemingly opposite effects [18]. in 100 μl lysis buffer (67 mM Tris, pH 8.8; 6.7 mM MgCl2; 0.5 mM β- Overexpression of PDGF-C or -D from the α-myosin heavy chain mercaptoethanol; 6.7 mM EDTA; 0.5% Triton-X100 and 500 μg/ml promoter (α-MHC) induces cardiac fibrosis in transgenic mice [19,20]. Proteinase K). The following PCR primers were used: Pdgfa fwd 5′- These mice were viable but developed hypertrophic hearts with signs of CTAAGGGATGGTACTGATTTTCGC-3′; Pdgfa rev 5′-AGGAATCTC dilated cardiomyopathy, proliferation of interstitial fibroblasts and GTAAATGACCGTCC-3′; Pdgfb fwd 5′-ATAGACCGCACCAACG- increased deposition of extracellular matrix. In addition, they devel- CCAACTTC-3′; Pdgfb rev 5′- AATAACCCTGCCCACACACTCTCC-3′. oped malformed vascular networks with decreased capillary density This resulted in a 411 bp product for both PDGF-Along and PDGF- and dilated blood vessels with increased α-smooth muscle actin Ashort, and 486 bp for PDGF-B. (ASMA) expression. Here, we have generated transgenic mice over- The PdgfraGFP/+ mice were genotyped by their strong GFP expressing PDGF-Ashort, PDGF-Along and PDGF-B from the same α- expression under a ultraviolet light, or with PCR using the following MHC promoter and phenotypically characterized their hearts. We also primers: 5′-CCCTTGTGGTCATGCCAAAC-3′;5′-GCTTTTGCCTCCA- analyzed the expression of PDGF receptors in developing and adult TTACACTG G-3′;5′-ACGAAGTTATTAGGTCCCTCGAC-3′ generating mouse